In situ polymerase chain reaction

ABSTRACT

The present invention concerns in situ polymerase chain reaction and provides methods and reagents for identifying cells containing at least one selected nucleic acid sequence which may be derived from the human immunodeficiency virus.

This application is a divisional application of U.S. patent applicationSer. No. 08/225,491 filed Apr. 11, 1994 now U.S. Pat. No. 5,589,333which is a continuation of U.S. patent application 07/829,921, nowabandoned, filed Feb. 3, 1992.

BACKGROUND OF THE INVENTION

Advances in nucleic acid research during the past decade haveconsiderably simplified the assays which utilize DNA and RNA probes forresearch and diagnostic purposes. Among various assays, the recentlyintroduced modification of the gene amplification method calledpolymerase chain reaction (PCR) qualifies as a technologicalbreakthrough. PCR in an in vitro gene amplification method whereby theDNA from a selected region of a genome can be amplified by more than amillion-fold in a few hours, provided that at least a portion of itsnucleotide sequences are already known. Segments of the gene sequencethat are at both sides of the portion of the gene which one wishes toamplify, are usually synthesized by an automatic DNA synthesizer. Thesetwo oligonucleotides, called "primers", are usually 10-30 base pairs(bp) long. The primers hybridize to opposite strands of DNA, and flankthe region of interest in the target DNA.

The PCR method has advantages over serological testing. For example, PCRhas been used to detect HIV-1 exposure in the seronegative sexualpartners of HIV-1-seropositive individuals, in HIV-1-seronegativeinfants and children, and in health care workers accidently exposed toHIV-1-positive blood or body fluids.

There are several disadvantages in the conventional DNA-PCR method. Dueto the sensitivity of the method, a small amount of contamination(usually from DNA fragments aerosolized during a previously performedamplification) may cause a false positive result. Current methods alsodo not permit determination of which cell types carry a certain gene.

The ability to identify individual cells, either latently orproductively infected, under the microscope would be extremely useful indelineating a latent state and emergence from it. This would be useful,not only in understanding the development of infection, but also as amore direct quantitative measure of the effects of antiviral therapy,and as an aid in understanding the mechanism of transmission of HIV-1.

Since the first description of human immunodeficiency virus type I(HIV-1) as the etiologic agent of AIDS, the numbers of cells infected inviva with HIV-1 isolated from individuals at various clinical stages ofdisease have been evaluated. These studies sought to correlate levels ofHIV-1 with disease pathogenesis and to determine the clinical course ofHIV-1-seropositive individuals.

The most detrimental clinical consequence of infection with HIV-1 is thesevere depletion of CD4-positive lymphocytes. It was assumed that suchdepletion was the result of selective infection and destruction ofCD4-positive lymphocytes by HIV-1. The studies of Harper et al., Proc.Natl. Acad. Sci. USA, 83:772-6 (1986), demonstrated that by using insitu hybridization for HIV-1-specific RNA, one can identify only1:10,000 to 1:100,000 peripheral blood mononuclear cells (PBMC) andlymph node cells positive for HIV-1 in vivo. These studies did notdiscount the possibility that HIV-1 may be present in a latent proviralform not expressing viral mRNA. The teaching of Harper et al. did noteliminate the possibility that HIV-1 selectively expressed only lowlevels of multiply-spliced RNA and did not express unspliced genomicHIV-1 RNA. The findings of Harper et al. taken with observations thatthe rate of isolation of HIV-1 from infected individuals was low,suggested that indirect mechanisms might mediate HIV-1-inducedCD4-positive lymphocyte destruction. Several mechanisms have beenproposed to account for the severe depletion of CD4-positive lymphocytesincluding: (1) production of direct cytopathic effect of HIV-1 onCD4-positive cells; (2) generation of HIV-1-specific cytotoxicT-lymphocytes (CTL) or antibody-dependent cellular cytotoxicity (ADCC),which destroy cells expressing surface HIV-1-specific proteins; (3)generation of giant cell syncytia formation, secondary to an interactionof the CD4 receptor and a fusion domain of the HIV-1 envelopeglycoproteins; and/or (4) production of antibodies against Tlymphocytes, bone marrow stem cells or immature thymocytes.

Recent data suggest a higher level of PBMC containing HIV-1-specific RNAin infected individuals. Studies utilizing limiting dilution assays haveshown that infectious HIV-1 can be isolated from an average of 1 in 400PBMC obtained from patients with AIDS, however, higher viral levels havebeen detected during acute HIV-1 seroconversion.

Since the introduction of Taq, the thermostable polymerase which broughtconvenience to the polymerase chain reaction (PCR) method, specialattention has been directed to the study of HIV-1 infection using thismethod. Modifications of the PCR method have been used to quantitativelyor semi-quantitatively assess the relative frequencies of HIV-1-infectedcells in PBMC, lymph nodes and other cell types. Schnittman et al.,Science, 245:305-8 (1989); Ann. Int. Med., 113:438-43 (1990), discloseusing a combination of cell sorting and quantitative DNA-PCR techniquesto observe that at least 1% of CD4-positive lymphocytes in patents withAIDS are infected with HIV-1. For patients who are HIV-1 asymptomatic,the levels of CD-4-positive lymphocytes was shown to be between 1:100and 1:100,000.

Spector et al., J. Infect. Dis., 164:4703-5 (1991), disclose using a"booster" PCR method and have calculated that at least 10% ofCD4-positive lymphocytes carry HIV-1 provirus in AIDS and symptomaticHIV-1-infected patients, whereas a relatively lower proportion ofCD4-positive lymphocytes are positive in HIV-1-seropositive individuals.

Studies utilizing a quantitative DNA PCR technique have suggested thatthere is a correlation between the clinical stage of HIV-1 infection andthe level of HIV-1-specific PCR signals.

A current limitation to PCR methodologies utilizing isolated DNA is thatone cannot directly associate the amplification results to a specificcell type or easily measure the percentage of cells which carry thetarget sequence. The HIV-1 virus has been demonstrated to infectCD4-positive lymphocytes, CD8-positive lymphocytes, monocytes,fibroblasts and glial cells in vivo. The CD4-positive lymphocyte isbelieved to be the primary reservoir for HIV-1 in the bloodstream andcells of the monocyte/macrophage lineage are believed to be the majorvirus reservoir in solid tissues. Therefore, it is highly desirable toidentify all cell types which carry the virus in vivo and determinewhich cells actively produce HIV-1.

Haase et al., Proc. Natl. Acad. Sci. USA, 87:4971-5 (1990), disclose thedevelopment of an in situ PCR methodology for Visna virus, a viralpathogen of sheep. Moreover, Haase et al. teach in situ PCR performed oncells infected with Visna virus in suspension. Haase et al. furtherdisclose that the reactions are performed using a Perkin-Elmer/Cetus DNAthermal cycler. Following the PCR reaction in solution, the contents ofthe tubes were centrifuged, resuspended and applied to a slide. ThePCR-amplified nucleic acid was fixed to the slide and hybridized to ¹²⁵I-labelled viral DNA for detection.

Oakes et al. (EP388171) teach purifying single-stranded targeted nucleicacid using non-porous, non-magnetic particles with complementary nucleicacid attached. Oakes et al. claim a method using standard PCRhybridization methodology and include a step of separating the hybridfrom the remainder of the specimen with a non-porous, non-magneticparticle. Oakes et al. indicate that the invention is useful sincepurified nucleic acids can be rapidly and simply purified. Oncepurified, the hybrids can be amplified by polymerase chain reaction.

Wang et al. (WO9102817) claim a method for quantifying a target nucleicacid segment in a sample. Wang et al. indicates that the invention isuseful for determining the quantity of specific RNA molecules in abiological sample.

Gyllensten and Erlich (WO9003444) teach a method for generatingsingle-stranded DNA by PCR that can be linked to an automatic sequencesystem for rapid sequence determination. The production ofsingle-stranded PCR products using limiting concentrations of one of thetwo primers is also disclosed.

Innis (WO9003443) teaches a method for structure-independentamplification of DNA by PCR using structure-destabilizing base analog inthe amplification reaction. Innis further teaches that the method isuseful to increase the specificity of PCR on nucleic acid templates thatcontain secondary structure and/or compressed regions.

Manos et al. (WO9002821) teach detecting human papilloma-virus usingconsensus primers in PCR to amplify particular genomic regions. Thepatent discloses a PCR reaction using at least a pair of primerscomplementary to separate strands of HPV DNA which hybridize to it andproduce an extension product.

Post et al. (WO9001547) teach isolation of thymidine kinase encoding DNAfrom herpes virus from degenerate primers and the production ofthymidine kinase negative feline herpes virus used to produce a livevaccine.

Erlich et al. (WO8911547) disclose HLA DP genotyping by amplifyingtarget DNA then hybridizing the amplified target to a panel ofsequence-specific oligonucleotides. It is disclosed that the method isespecially useful for assessing the risk of autoimmune disease.

U.S. Pat. No. 5,008,182 (Kwork et al.) discloses the detection ofAIDS-associated virus by PCR in free solution or after immobilization ona solid support.

Until the present invention, there have been no methods which allow insitu PCR amplification of specific DNA fragments in intact cells.

SUMMARY OF THE INVENTION

The present invention is an in situ PCR method that can be utilized toamplify selected genetic regions in intact cells. To that end, thepresent invention provides methods for identifying cells containing atleast one selected nucleic acid sequence comprising: (a) fixing cells toa solid support; (b) carrying out a polymerase chain reaction bycontacting cells and a selected nucleic acid sequence or sequences witholigonucleotide primers complementary to regions of the selected nucleicacid sequence to form a hybrid and generating an extension product; (c)separating the extension product generated in step (b) to providesingle-stranded nucleic acid molecules of the selected nucleic acidsequence; (d) repeating steps (b) and (c) to amplify said selectednucleic acid sequence; (e) hybridizing labelled probe to at least oneamplified nucleic acid sequence; and (f) observing labelled cells. Thepresent invention may have application in the diagnosis of HIV-1infection in individuals and for the determination of the prognosis ofAIDS patients. In a preferred embodiment the present invention can beused to determine the proportion of PBMC carrying HIV-1 provirus andproviral sequences isolated from infected individuals. In addition, thisinvention has broad application and can be applied for identification ofessentially any gene(s); bacterial, viral, fungal, parasitic, aberrantgenes, oncogenes, normal human genes, genetic defects, genetic markers(i.e., ALA haplotypes, other transplantation genes, blood groups andsubtypes, rare genetic events (like translocation of genes), cancermarkers, presence of certain genetic predilections (i.e., Alzheimerdisease, cystic fibrosis, etc.) and many others.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic representation of a preferred embodiment of anapparatus for carrying out the methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention intact fixed cells function as amplificationvessels. Once cells are heat-fixed and permeabilized byparaformaldehyde, PCR reagents, including Taq DNA polymerase and PCRprimers, are allowed to diffuse into the cell. Following amplificationsteps, most of the amplified PCR product remains within the nucleus ofthe cell due to certain favorable conditions provided by the presentinvention. It is believed that the amplified PCR product remains boundto either the nuclear double membranes and nuclear matrix, or existswithin the cytoplasmic membrane.

The present invention provides methods for identifying cells containingat least one selected nucleic acid sequence comprising: (a) fixing cellsto a solid support by exposing the cells to dry heat, for example; (b)carrying out a polymerase chain reaction by contacting cells andselected nucleic acid sequence or sequences with oligonucleotide primerscomplementary to regions of the selected nucleic acid sequence orsequences to form a hybrid and generating an extension product; (c)separating the extension product generated in step (b) from the templateto provide single-stranded nucleic acid molecules of the selectednucleic acid sequence or sequences; (d) repeating steps (b) and (c) toamplify said selected nucleic acid sequence; (e) hybridizing labelledprobe to at least one amplified nucleic acid sequence; and (f) observinglabelled cells. At least one of the selected nucleic acid sequence orsequences derive from a human virus. In the invention, at least one ofthe selected nucleic acid sequence or sequences derived from a humanvirus may be derived from recombinants, mutants or variants of suchhuman virus. The present invention also provides methods whereby atleast one of the selected nucleic acid sequences derived from a humanvirus are derived from a human retrovirus. In a preferred embodiment ofthe present invention, at least one of the selected sequences derivefrom human immunodeficiency virus, and comprise one of the followingsequences: long terminal repeat (LTR) sequence; the tat gene sequence;the gag gene sequence; the pol gene sequence or the env gene sequence.Methods of the present invention further contemplate that at least oneof the sequences utilized in the polymerase chain reaction derived fromhuman immunodeficiency virus are proviral nucleic acid sequences.Methods using oligonucleotide primers derived from sequencescomplementary to sequences of HIV-1 or HIV-2 virus are alsocontemplated. Furthermore, the present invention provides methodswhereby at least one of the sequences complementary to the sequences ofHIV-1 or HIV-2 is derived from recombinants, mutants or variants ofHIV-1 or HIV-2.

The present invention uses probes that serve as internal controls in thepolymerase chain reaction. Control probes may be complementary to eitherchromosomal or episomal targeted nucleic acids and serve as positivecontrols for the polymerase chain reaction; control probes may not becomplementary to either chromosomal or episomal targeted nucleic acidsand serve as a negative controls for the polymerase chain reaction. In apreferred embodiment, oligonucleotide primers utilized in the polymerasechain reaction comprise nucleic acid sequences complementary to HLA-DQαor SK-19; HLA-DQα or SK-19 probes may be as internal controls in a mixedprobe reaction comprising probes complementary to other targetsequences.

Methods of the present invention provide that cells utilized in the insitu polymerase chain reaction may be obtained from mammalian tissues,particularly but not limited to human tissues. More specifically, thesetissues may be obtained as diagnostic specimens. Further, the cells maybe obtained from tissue aspirates and bodily fluids. Cells that aretransformed, hyperplastic or obtained from carcinomas are alsocontemplated to be useful in the methods of the present invention. In apreferred embodiment, cells used in the polymerase chain reaction arehuman blood cells, particularly but not limited to human peripheralblood monocytic cells; human peripheral blood monocytic cells utilizedin methods of the present invention are human blood cells infected withhuman immunodeficiency virus, including HIV-1 and HIV-2.

In the present invention, solid supports capable of binding cells areemployed; teflon is preferred. In a more preferred embodiment, a solidsupport comprises teflon and glass. Solid supports may be in the form ofmicroscope slides comprising teflon and glass.

To demonstrate the invention, in situ PCR was performed by mixinglatently HIV-1-infected U1 monocytoid cells, Folks et al., Science,238:800-2 (1987), with HIV-1-uninfected U937 monocytoid cells, Sundstromet al., Int. J. Cancer, 17:565-77 (1976), in various proportions. PCRprimers specific for, and complementary to, selected sequences, wereused. Sequences used for demonstration of the methods of the presentinvention were derived from HIV-1 and the HLA-DQα gene. It wasdemonstrated using microscopy, that U1 cells subjected to in situ PCRare all positive for the HIV-1 provirus. Uninfected U937 cells subjectedto the same procedure did not demonstrate any HIV-1-positive cells.There was a 1:1 ratio of U1 to U937 cells. In situ PCR resultsdemonstrated infected cell ratios of 1:10 and 1:100 respectively. Thisconcurs with reports which demonstrate that the U1 cells are a latentlyHIV-1-infected subclone of U937 cells having 2 HIV-1 proviral copies percell. HLA-DQα primers used as a positive control demonstrated positiveamplification in all cells following hybridization with a biotinylatedprobe complementary to HLA-DQα.

Sensitivity of the in situ PCR methods of the present invention wasdemonstrated using ACH-2 cells, an HIV-1 latently-infected subclone ofthe T-lymphocytic cell line, CEM, which contains only one HIV-1 proviruscopy per cell. See Clouse et al., J. Immunol., 142:431-8 (1989). ACH-2cells subjected to in situ PCR were all hybridization-positive and atthe same degree of intensity. These cells were mixed in varyingproportions with uninfected CEM cells and hybridization was demonstratedby microscopy.

Using the methods of the present invention it has been demonstrated thatin situ amplification can be efficiently carried out in cell populationswhich are known to carry one or two copies of HIV-1 provirus per cell.Modifications of gene amplification methods of the present inventionmakes in situ PCR several-fold more sensitive than standard DNA-PCR. Ithas further been demonstrated that amplified DNA of the presentinvention does not leak out of infected cells and contaminate uninfectedcells. This is a surprising result that renders the methods of thepresent invention useful as sensitive in situ PCR diagnostic assays.

Proportions of PBMC containing HIV-1 provirus were demonstrated over abroad ratio range using in situ PCR methods of the present invention.Table I illustrates the results from the quantitation of HIV-1 provirusin HIV-1 seropositive individuals. Uninfected individuals were used asnegative controls. PBMC were isolated from patients, bound to solidsupports by fixing, and permeabilized. The in situ PCR analysis wascarried out as described for the tissue culture cells, using primersspecific for HIV-1. In Table I, a single asterisk indicates that eachpatient's PBMC obtained from a single phlebotomy were evaluated at leasttwice using the in situ PCR technique. Double asterisks denote that allfive individuals in Stage IV D had Kaposi's Sarcoma and no lymphomas.The letters "ND" indicate that these data have not been determined.Disease stages are indicated by Roman numerals and are described brieflyfollowing each designation. The arithmetic mean percentage ofHIV-1-positive PBMC is expressed with the standard deviation.

                  TABLE I                                                         ______________________________________                                        Quantitation of HIV-1 Provirus in HIV-1                                       Seropositive Individuals                                                                                       Total                                        Modified             Percentage of                                                                             CD4-Positive                                 CDC-HIV     Patient  HIV-1-Positive                                                                            T Lymphocyte                                 Classification                                                                            (Coded)  PBMC*       Counts (per mm.sup.3)                        ______________________________________                                        HIV-1-seropositive                                                                        MIC      3.3 ± 1.3                                                                              339                                          Asymptomatic                                                                              STA      2.7 ± 1.7                                                                              256                                          (Stage II)  NOV      0.15 ± 0.3                                                                             ND                                                       BON      0.66 ± 0.2                                                                             ND                                                       SAY      0.5 ± 0.01                                                                             ND                                                       FLE      0.36 ± 0.2                                                                             ND                                                       SVM      0.8 ± 0.25                                                                             ND                                                       BOT      0.9 ± 0.1                                                                              ND                                                       ROY      0.09 ± 0.02                                                                            ND                                                       BER      1.6 ± 1.0                                                                              ND                                                       HER      3.6 ± 2.0                                                                              ND                                                       DUG      1.1 ± 0.1                                                                              ND                                                       ADA      0.2 ± 0.06                                                                             ND                                                       MAU      0.2 ± 0.07                                                                             ND                                                       X        0.3 ± 0.02                                                                             349                                                      Y        0.3 ± 0.05                                                                             612                                                      Z        0.3 ± 0.06                                                                             429                                                      AA       0.1 ± 0.05                                                                             1050                                                     BB       0.3 ± 0.07                                                                             572                                          HIV-1-seropositive                                                                        EVA      5.4 ± 1.4                                                                              28                                           Persistent  SED      2.8 ± 1.8                                                                              338                                          Generalized ROB      12.0 ± 1.8                                                                             657                                          Lymphadenopathy                                                                           ANT      10.7 ± 2.0                                                                             168                                          (Stage III) PAD      11.8 ± 1.9                                                                             584                                                      CHR      13.5 ± 2.1                                                                             ND                                                       TTI      5.3 ± 1.5                                                                              ND                                                       BOR      6.3 ± 1.5                                                                              ND                                                       ASD      4.0 ± 2.2                                                                              ND                                                       D        1.2 ± 0.5                                                                              21                                                       N        7.5 ± 0.6                                                                              84                                                       R        1.3 ± 0.5                                                                              39                                                       T        4.0 ± 1.0                                                                              336                                          AIDS        PAR      11.2 ± 1.4                                                                             164                                          (Stages IV A-C)                                                                           POP      8.0 ± 1.6                                                                              107                                                      GRE      7.8 ± 1.3                                                                              226                                                      LAN      11.8 ± 1.9                                                                             104                                                      A        0.13 ± 0.06                                                                            5                                                        C        1.5 ± 0.7                                                                              30                                                       E        1.5 ± 0.7                                                                              132                                                      F        2.1 ± 0.14                                                                             3                                                        G        0.5 ± 0.07                                                                             6                                                        H        0.2 ± 0.05                                                                             33                                                       I        7.3 ± 2.0                                                                              80                                                       L        8.5 ± 0.7                                                                              144                                                      M        5.0 ± 1.0                                                                              32                                                       O        8.0 ± 1.0                                                                              188                                                      P        4.0 ± 0.5                                                                              180                                                      Q        3.0 ± 1.0                                                                              39                                                       S        4.7 ± 0.5                                                                              448                                                      V        1.0 ± 0.2                                                                              13                                                       W        2.0 ± 0.1                                                                              63                                           AIDS        DOM      1.8 ± 0.7                                                                              22                                           (Stage IV D)**                                                                            DWI      2.0 ± 0.89                                                                             290                                                      BUL      2.0 ± 0.89                                                                             20                                                       B        0.8 ± 0.1                                                                              25                                                       K        1.2 ± 0.2                                                                              14                                           ______________________________________                                    

The percentage of HIV-1 positive PBMC varied between 0.09% and 13.5%, asshown in Table 1. Nineteen asymptomatic HIV-1-seropositive individualsexhibited a range of 0.09% to 3.6%. Thirteen other HIV-1 infectedpatients having persistent generalized lymphadenopathy (Stage III), ascategorized by the modified CDC HIV-classification system, exhibited1.3% to 13.5% HIV-1 provirus positive cells. Four of these thirteenindividuals (N,R,EVA and ANT) developed oral candida infection (thrush)in addition to persistent generalized lymphadenopathy. Individuals inStages IV A, B, and C revealed 0.13% to 11.8% of PBMC positive for HIV-1provirus. By contrast, patients in Stage IV D, demonstrating evidence ofKaposi's Sarcoma (KS), showed relatively low percentages of PBMCpositive for HIV-1 provirus ranging from 0.8% to 2.0%. Thus, PBMC fromindividuals who were in CDC Stage II and were asymptomatic showedrelatively low percentages of HIV-1 positive PBMC, as compared to StagesIII or IV A-C (p<0.001), as determined by the Student's t test. PBMCfrom patients classified as CDC Stage IV D (patients with Kaposi'sSarcoma but without opportunistic infection) also exhibited a relativelylow percentage of cells infected with HIV-1 (p<0.08). No statisticallysignificant difference in the level of HIV-1 positive PBMC was notedcomparing individuals in Stage III versus Stage IV A-C. All elevenHIV-1-seronegative controls were consistently negative by in situ PCR.

Evaluation of the same PBMC from HIV-1-seropositive individualsutilizing standard in situ hybridization techniques revealed only 1 in5×10³ to 1×10⁵ PBMC positive for HIV-1-specific nucleic acids. Theseresults were consistently observed utilizing gag, tat and LTR probes andprimers. Thus, the vast majority of HIV-1-infected PBMC in vivo do notexpress large quantities of HIV-1-RNA or are actively producing highlevels of virus.

The ability to detect a significantly higher level of HIV-1-infectedcells using in situ PCR of the present invention as compared to usingother techniques, such as viral culture or standard DNA-PCR, is based onthe exquisite sensitivity of this technique. It is believed that areason for the unusually high sensitivity of the in situ PCR methods ofthe present invention as compared to the standard DNA-PCR method is thatthere is no dilution of HIV-1 into non-HIV-1 containing DNA. Suchdilution lowers PCR sensitivity. In the in situ PCR methods provided, acell is believed to be an ultra-small amplification container, whereamplification of a DNA segment can be carried out in a concentratedfashion and without dilution with other DNA.

The large numbers of provirus-positive PBMC in the blood ofHIV-1-infected individuals suggests that some of these proviruses may betranscriptionally quiescent or latent in vivo. This demonstration ofproviral latency in vivo is pertinent to the understanding of HIV-1pathogenesis. A molecular mechanism of HIV-1 proviral latency has beendescribed although latent infection prior to proviral integration mayalso exist.

The demonstration of significantly higher numbers of PBMC harboringHIV-1 provirus obtained using the methods of the present invention, ascompared to levels of infectious HIV-1 per PBMC in co-culture assays,indicates that some HIV-1 proviral copies may either be defective ormaintained in cells not activated to produce virions in cell cultures.Defective HIV-1 proviral copies have been demonstrated in vivo.Therefore, the evaluation of proviral latency and defective viralgenomes in vivo remain important areas in the study of the complexpathogenesis and natural history of clinical HIV-1 infection. Theability to precisely measure HIV-1 proviral load in the peripheral bloodin vivo is critical to evaluation of the clinical efficacy oftherapeutic interventions and as a prognostic indicative of HIV-1disease progression.

In situ PCR methods of the present invention have allowed quantitationof the percent of cells positive for HIV-1 provirus and have shown arelationship to the stage of HIV-1 clinical infection. This observationhas important implications in the determining the prognosis of apatient's disease. Patients in CDC Stage II had a significantly lowerpercentage of HIV-1-positive PBMC than those in Stage III and Stages IVA-C. Patients in Stage IV D (KS only) had relatively low numbers ofHIV-1-infected cells. This finding is rather surprising, and it may beone of the reasons that some investigations have failed to observe thecorrelation between the clinical stage of HIV-1 infection and the degreeof DNA-PCR amplification. This may also account for the longer life spanof patients with KS, as compared to other patients with AIDS.

One of the main concerns of investigators utilizing the standard DNA-PCRmethod is false positive results due to contamination of samples byHIV-1-positive amplified genetic segments. However, the use of in situPCR methods of the present invention greatly diminishes such concernssince it is believed that contamination of amplified genetic segmentswill contaminate only a few cells. It is believed that these fewcontaminated cells will not provide totally false positive results as isthe case for standard DNA-PCR methods where combined amplificationproducts are measured in toto.

In situ PCR methods provided by the present invention, which allowamplification of specific DNA fragments in intact cells, have greatpotential for determining the actual peripheral blood proviral load invarious stages of HIV-1 infection and in evaluating the efficacy ofvarious therapeutic interventions. In addition, there is currently noreliable way to determine the states of HIV-1 infection in infants bornto HIV-1-seropositive mothers, soon after birth. Methods in the presentinvention may be useful to identify such infected infants. It is alsobelieved that the methods of the present invention could be utilized toidentify contaminated donated blood form individuals who have not yetseroconverted.

Another aspect of the present invention is that the methods disclosedherein lend themselves to automation and, accordingly, the presentinvention provides apparatus for performing in situ PCR. Referring toFIG. 1, there is shown a schematic representation of an apparatus madein accordance with the present invention. In a most preferredembodiment, specially designed slides 10 that have two wells formedtherein are used as cell containers. Cells or tissue sections to betested in a specific gene or genes are placed in the wells in the slides10. These cells or tissue specimens are then sequentially processed inone or more separate chambers or other apparatus. Preferably, as shownin FIG. 1, the slide 10 is placed upon a transfer means such as aconveyor belt 100 that moves the slide 10 to the various section of theapparatus that are discussed in detail below. It should be understood,however, that the sequence of operations set forth herein is exemplaryand neither the combination of apparatus nor the steps illustrated anddescribed are meant to limit the scope of this aspect of the presentinvention. Moreover, although the description below is directed towarddescribing the processing of a single slide, it will be understood thata plurality of slides can be processed together and that certainoperations may be performed in parallel upon different batches ofslides.

In a preferred embodiment of the apparatus of the present invention aslide 10 (or a plurality of slides 10) enters an oven 200 for heatfixating, preferably at about 105° C. for about 90 seconds. The slide isthen placed in a paraformaldehyde treatment apparatus 202 at atemperature of about 37° C. for about two hours. Those of ordinary skillwill realize that the time and temperature requirements of the processdictate that this section of the apparatus will be thermally controlled.

After the paraformaldehyde treatment is completed, the slide 10 istransferred to a washing station 204 that most preferably washes theslide three times with a phosphate buffered saline (PBS) washingsolution for about 10 minutes. Two consecutive single 10 minute PBSwashes follow the first triple wash. Although the washing station 202 isillustrated as a single element of the apparatus, it will be understoodthat numerous configurations comprising several washing stations couldbe used to facilitate throughput. After the washings are completed, theslide 10 is moved to a proteinase K (pk) treatment apparatus 206 andtreated at 55° C. for up to three hours, depending on the type ofspecimen contained in the slide 10. As mentioned above, the elevatedtemperature at which the proteinase K treatment is carried out requiresthat the apparatus comprise a thermally controlled (heated) zone.

The slide 10 is then transferred to a washing station 208 and is mostpreferably single washed using a PBS solution. The washing also resultsin the heat inactivation of the proteinase K. Although the washingstation 208 is depicted as a separate portion of the apparatus of thepresent invention, it should be understood that the washing station 202or another single wash station could be implemented by routing thetransfer means 100 differently. In other words, the embodimentillustrated in FIG. 1 is constructed as a linear array and thus, aseparate washing station 208 is shown. The slide is then take from thewashing station 208 and transferred to buffer apparatus 210 where PCRbuffers, primers, nucleotides and heat stable polymers are added to theslide walls to form a "cocktail." For the purpose of the presentinvention, "buffering apparatus" therefore is adopted to add one or moreof these to the cells.

The slide 10 containing this cocktail is then thermally cycled bytransferring it into an oven 212. As was the case with the washingstations 204, 210, the oven 212 need not be a separate piece ofapparatus. As illustrated, the slide 10 could instead be transferredback to the oven 200 in which the heat fixation described above wasperformed. In any event, thermocycling is preferably undertaken forabout thirty cycles between temperatures of 95° C., 42° C., and 72° C.,for 1 minute each. However, temperature and cycle time may varyaccording to primers and DNA to be amplified.

After thermal cycling, the slide 10 is again transferred to a washstation 214 which is again most preferably a separate single wash PBSstation 214. After the wash is completed, the slide 10 is transferred toa chamber containing the appropriate apparatus to effect in situhybridization with specific and non-specific probes. Thus, after thisstep is complete, the slide 10 is transferred to a means for developingthe color in the slides 218 using techniques well known to those ofskill in the art. The developed slides are then dried and mounted topermit them to be read using either light microscopy or image analysis.

The embodiment of the apparatus illustrated in FIG. 1 is based upon theconcept of a linear "production line" that would be able to process aplurality of slides in an automated fashion. However, as well known tothose familiar with laboratory automation, the implementation of such afixed production line requires a significant investment of both time andmoney. Moreover, the resulting apparatus is relatively difficult toadapt, should the processing steps be modified. Thus, in certaininstances, it may be desirable to implement the apparatus of the presentinvention by using a flexible automation element such as a programmablerobotic manipulator that has the various "stations" or chambersdescribed above arrayed around the manipulator, within its reachenvelope. Such a flexible system would admit to numerous variations andmay permit the elimination of certain of the duplicate elements shown inFIG. 1. Those of skill in the art of designing such equipment couldprogram a robot and design interfaces to permit the methods of thepresent invention to be carried out in an effective manner.

Having generally described the invention, a more complete understandingcan be obtained by reference to the following examples which areprovided for purposes of illustration only and are not intended to belimiting.

EXAMPLES Example 1 Obtaining and Processing Blood Specimens

Blood specimens were obtained from 56 HIV-1-seropositive adults.Nineteen patients were asymptomatic, while thirty-seven weresymptomatic. Heparinized peripheral blood specimens were obtained formeach individual and assigned a code, then forwarded for laboratoryanalysis. Blood specimens were also drawn from eleven HIV-1-seronegativeindividuals. Identity and HIV-1 serological status remained unknown tothe MRL, until the completion of the studies.

Blood samples were processed within 4 hours after venipuncture and PBMCwere isolated by Ficoll-Hypaque gradient centrifugation. Cells werewashed twice with phosphate-buffered saline (PBS) and placed on slidesfor in situ PCR, as described below.

Example 2 In Situ Polymerase Chain Reaction

To perform in situ PCR for detection of HIV-1 provirus, cells (1×10⁵cells per ml) were seeded into the wells of specially designed heavyteflon coated (HTC) slides (Cell-line Associates, Inc. Newfield, N.J.08344 CAT #10-12, 14 mm wells). Slides were air dried and the placedsequentially, first on a heat block at 105° C. for 90 seconds and thenin 1% paraformaldehyde-phosphate-buffered saline (PBS) solution (pH 7.4)for 1 hour. Paraformaldehyde was inactivated by washing the slides in 3X PBS, then the slides were washed 3 times in 1 X PBS. Endogenousperoxidase activity was removed by quenching the specimens with a 3%solution of hydrogen peroxidase, overnight, at 37° C. The slides werethen treated with Proteinase K (60 μg/ml in PBS) for 2 hours at 55° C.Proteinase K was inactivated by placing the slides on a heat block at90° C. for 2 minutes and, finally, the slides were washed in distilledwater and air dried.

The cells were then subjected to amplification. A primer pair,complementary to conserved regions of HIV-1 gag (SK38; nucleotides:1551-1578; SK39; nucleotides: 1638-1665, Synthetic Genetics, San Diego,Calif.), was used for amplification of HIV-1-DNA. Fifteen μl of aPCR-reaction mixture containing 10 μM of dATP, dCTP, dGTP and dTTP, 20μM of each primer, 50 mM KCL, 10 mM Tris (pH 8.3), 2.5 MM MgCl₂ and 1.0μl Taq polymerase (1 U/μl, Gene Amp, Cetus), was added to the top twowells of each slide, whereas the bottom well received a PCR-mixturelacking the primers. These slides were covered with 22×60 mm coverslips.Coverslips were sealed with a clear nail polish. Slides were placed onan automatic thermocycler (M.J. Research Boston, Mass.) andamplification was carried out at 94° C./45° C./72° C. for 1 minute each,for 30 cycles. The primers for HLA-DQα (HLA-DQ-GH-26/27, SyntheticGenetics), with a biotinylated probe for HLA-DQα, were used as positivecontrols.

Example 3 Probing In Situ PCR Extension Products

After amplification, all slides were washed in 2 X SSC buffer (0.3M NaCland 0.03M Na-citrate) and amplification products were detected by abiotinylated oligonucleotide (SK 19, nucleotides: 1595-1635: SyntheticGenetics), utilizing the in situ hybridization method. Hybridizationmixture contained 15-25 pg of biotin-labelled probe, 10 mM DTT, 2 X SSC,fragmented salmon sperm DNA (1 mg/ml), 50% formaldehyde, 2% bovine serumalbumin, and E. coli transfer-RNA (1 mg/ml). The mixture was applied toeach well of the slide. Slides were sealed with coverslips and incubatedon a heat block at 92° C. for 5 minutes. Slides were then transferred toanother heated humidified chamber at 48° C. for 4 hours. These slideswere thoroughly washed with PBS and then incubated with streptavidinperoxidase complex (100 μg/ml in PBS, pH 7.2) for 1 hr at 37° C. Afterincubation, slides were thoroughly washed with phosphate buffered saline(PBS). The color was developed with 3' amino 9' ethylene carbozone (AEC)in the presence of 0.03% hydrogen peroxide in 50 mM acetate buffer (pH5.0) for 10 minutes at 37° C. The slides were then washed and coverslipswere applied, with a 50% solution of glycerol/PBS. Slides were analyzedusing optical microscopy.

In all amplifications, one slide well was used as an internal control,in which amplified cells were hybridized with a unrelated probe.Hybridization with an unrelated probe (HLA-DQα) gave negative results,whereas with a related probe (SK-19), the technique yielded the expectedresults. An HIV-1-specific probe (tat gene probe) complementary to aregion of HIV-1 not amplified by the gag primers yielded very fewpositive cells. Other HIV-1-specific sets of primers and probes (longterminal repeat (LTR) and tat) gave consistent results in the assaysystem of the present invention. U1 and ACH-2 cells evaluated usingcytomegalovirus-specific primers and probes yielded no detectablepositively staining cells.

Example 4 Statistical Analyses

The arithmetic mean percentage of HIV-1-positive PBMC determined usingthe methods of the present invention is expressed with the standarddeviation. The Student's t-test was used to compare differences inHIV-1-infected PBMC among different groups of individuals whose PBMCwere analyzed.

What is claimed is:
 1. A method for identifying a cell containing atleast one copy of a selected nucleic acid sequence comprising:(a)exposing said cell to dry heat thereby fixing said cell to a solidsupport to produce a heat fixed immobilized cell; (b) contacting saidheat fixed immobilized cell with paraformaldehyde to produce aparaformaldehyde treated, heat fixed immobilized cell; (c) contactingsaid paraformaldehyde treated, heat fixed immobilized cell containingselected nucleic acid sequence or nucleic acid sequences witholigonucleotide primers complementary to regions of selected nucleicacid sequence or sequences to form a nucleic acid hybrid and generatingan extension product by amplification in said cell; (d) separating intosingle stranded nucleic acid molecules, the extension product generatedin step (c) to provide single-stranded nucleic acid molecules of theselected nucleic acid sequence or sequences in said cell; (e) repeatingsteps (c) and (d) to amplify said selected nucleic acid sequence orsequences; (f) hybridizing a labeled nucleic acid probe to saidamplified selected nucleic acid sequence or sequences provided in step(e); and (g) observing a labeled cell produced in step (f), wherein alabeled cell indicates a cell containing at least one copy of a selectednucleic acid sequence.
 2. The method of claim 1 wherein at least one ofthe selected nucleic acid sequences derive from a human virus.
 3. Themethod of claim 2 wherein at least one of the selected nucleic acidsequences derive from human immunodeficiency virus and comprise one ofthe following sequences: long terminal repeat (LTR) sequence; the tatgene sequence; or the gag gene sequence.
 4. The method of claim 2whereby at least one of the selected nucleic acid sequences are proviralnucleic acid sequences.
 5. The method of claim 1 wherein saidoligonucleotide primers are derived from sequences complementary tosequences of HIV-1 or HIV-2.
 6. The method of claim 2 wherein at leastone of said nucleic acid sequences is derived from recombinants, mutantsor variants of said human virus.
 7. The method of claim 1 wherein saidoligonucleotide primers are derived from sequences complementary toHLA-DQα or SK-19.
 8. The method of claim 1 wherein said cells are humancells.
 9. The method of claim 8 wherein said cells are human peripheralblood lymphocytic cells.
 10. A method for identifying a cell containingat least one copy of a selected nucleic acid sequence comprising:(a)exposing said cell to dry heat thereby fixing said cell to a solidsupport; (b) paraformaldehyde treating said heat fixed cell; (c)contacting said heat fixed, paraformaldehyde treated cell containingselected nucleic acid sequence or nucleic acid sequences with a singleprimer pair complementary to regions of selected nucleic acid sequenceor sequences to form a nucleic acid hybrid and generating an extensionproduct by amplification in said cell; (d) separating into singlestranded nucleic acid molecules, the extension product generated in step(c) to provide single-stranded nucleic acid molecules of the selectednucleic acid sequence or sequences in said cell; (e) repeating steps (c)and (d) to amplify said selected nucleic acid sequence or sequences; (f)hybridizing a labeled nucleic acid probe to said amplified selectednucleic acid sequence or sequences provided in step (e) in said cell;and (g) observing a labeled cell produced in step (f), wherein saidlabeled cell indicates a cell containing at least one copy of a selectednucleic acid sequence.
 11. The method of claim 10 wherein saidoligonucleotide primers are fully complementary to regions of selectednucleic acid sequence or sequences that consist of at least 10nucleotides.
 12. The method of claim 11 wherein said oligonucleotideprimers are fully complementary to regions of selected nucleic acidsequence or sequences that comprise 10-30 nucleotides.
 13. The method ofclaim 1 wherein said oligonucleotide primers are fully complementary toregions of selected nucleic acid sequence or sequences that consist ofat least 10 nucleotides.
 14. The method of claim 13 wherein saidoligonucleotide primers are fully complementary to regions of selectednucleic acid sequence or sequences that comprise 10-30 nucleotides. 15.The method of claim 10 wherein said amplification comprises thepolymerase chain reaction.
 16. The method of claim 1 wherein saidamplification comprises the polymerase chain reaction.